Method and apparatus for automated spectral calibration

ABSTRACT

The disclosure generally relates to a method and apparatus for automated spectral calibration of a spectroscopy device. In one embodiment, the disclosure relates to a method for simultaneous calibration and spectral imaging of a sample by: simultaneously illuminating the sample and a calibrant with a plurality of illuminating photons; receiving, at the spectrometer, a first plurality of photons collected from the sample and a second plurality of photons collected from the calibrant; forming a calibrant spectrum from the first plurality of collected photons and a sample spectrum from the second plurality of collected photons; comparing the calibrant spectrum with a reference spectrum of the calibrant to determine a wavelength-shift in the calibrant spectrum; applying the wavelength-shift to the sample spectrum to obtain a calibrated sample spectrum.

The instant disclosure claims the filing-date benefit of ProvisionalApplication No. 60/750,784 filed Dec. 16, 2005, and ProvisionalApplication No. 60/754,720 filed Dec. 29, 2005. The disclosure of eachof these applications is incorporated herein in its entirety.

BACKGROUND

Conventional spectroscopic imaging systems are generally based on theapplication of high resolution, low aberration lenses and systems thatproduce images suitable for visual resolution by the human eye. Theseimaging systems include both microscopic spectral imaging systems aswell as macroscopic imaging systems and use complex multi-element lensesdesigned for visual microscopy with high resolution aberrationsoptimized for each desired magnification.

Spectroscopic imaging combines digital imaging and molecularspectroscopy techniques, which can include, Raman scattering,fluorescence, photoluminescence, ultraviolet, visible and infraredabsorption spectroscopies. When applied to the chemical analysis ofmaterials, spectroscopic imaging is commonly referred to as chemicalimaging. Instruments for performing spectroscopic (i.e. chemical)imaging typically comprise image gathering optics, focal plane array(FPA) imaging detectors and imaging spectrometers.

The choice of FPA detector is governed by the spectroscopic techniqueemployed to characterize the sample of interest. For example, silicon(Si) charge-coupled device (CCD) detectors, a type of FPA, are typicallyemployed with visible wavelength fluorescence and Raman spectroscopicimaging systems, while indium gallium arsenide (InGaAs) FPA detectorsare typically employed with near-infrared spectroscopic imaging systems.A variety of imaging spectrometers have been devised for spectroscopicimaging systems. Examples include, without limitation, gratingspectrometers, filter wheels, Sagnac interferometers, Michelsoninterferometers and tunable filters such as acousto-optic tunablefilters (AOTFs) and liquid crystal tunable filters (LCTFs).

The efficiency of the of imaging spectrometers is also a function of thesystem-specific noise caused by background light, room temperature, thewavelength of the scattered light and the electro-mechanical or opticalintangibles associated with the spectrometer. For example, the LCTF hasa wavelength dependent transmission modulation which affect's theaccuracy and the efficiency of measuring sharp Raman bands with weakRaman scatterers. Experiments with certain LCTF devices show complicatedinteractions arising in the material and structure of the imagingdevices produce a spatial and spectral modulation of light comingthrough the imaging device. The modulation produces an apparentbackground signal that is not uniform and masks the real signal.

Virtually all spectral imaging devices depend on the optical propertiesand transmission of light through one or more optical devices in orderto produce the desired filtering effect. Such devices also have complexinternal configuration which affects transmission of light through thedevice. Although the imaging filters are designed to minimize suchaberrations, residual effects remain which limit the accuracy of thedevice and requiring the additional step of calibration prior to imagingthe sample. However, implementing such sequential steps duringexamination of certain in vivo biological samples is inefficient,impractical and at times, impossible.

SUMMARY

In one embodiment, the disclosure relates to a method for simultaneouscalibration and spectral imaging of a sample comprising: simultaneouslyilluminating the sample and a calibrant with a plurality of illuminatingphotons; receiving, at the spectrometer, a first plurality of photonscollected from the sample and a second plurality of photons collectedfrom the calibrant; forming a calibrant spectrum from the firstplurality of collected photons and a sample spectrum from the secondplurality of collected photons; comparing the calibrant spectrum With areference spectrum of the calibrant to determine a wavelength-shift inthe calibrant spectrum; applying the wavelength-shift to the samplespectrum to obtain a calibrated sample spectrum.

In another embodiment, the disclosure relates to a system forsimultaneous calibration and dispersive and/or spectral imaging of asample comprising: an input for simultaneously receiving a firstplurality of photons collected from the sample and a second plurality ofphotons collected from a calibrant; a spectrograph for forming a samplespectrum from the first plurality of photons and a calibrant spectrumfrom the second plurality of photons; a first processor for comparingthe calibrant spectrum with a reference spectrum of the calibrant todetermine a wavelength-shift in the calibrant spectrum; and a secondprocessor for applying the wavelength-shift to the sample spectrum toobtain a calibrated sample spectrum.

In still another embodiment, the disclosure relates to an apparatus forsimultaneous calibration and dispersive and/or spectral imageacquisition of a sample, comprising: a processing circuit forsimultaneously receiving a calibrant spectrum and a sample spectrum, anda memory in communication with the processing circuit, the memorystoring instructions for the processing circuit to: (i) process thecalibrant spectrum to locate and identify a plurality of peaks, (ii)compare the plurality of peak locations in the calibrant spectrum with aplurality of corresponding peak locations in a reference spectrum of thecalibrant, and (iii) determine a wavelength-shift as a function of acomparison between at least one peak location in the calibrant spectrumand a corresponding peak location in the reference spectrum; calibratethe sample spectrum by applying the wavelength-shift to the samplespectrum.

In another embodiment, the disclosure relates to a method forsimultaneous calibration and imaging of a sample in a spectrometer, themethod comprising: simultaneously illuminating the sample and anintrinsic calibrant with a plurality of illuminating photons; receiving,at the spectrometer, a first plurality of photons collected from thesample and a second plurality of photons collected from the intrinsiccalibrant; forming a sample spectrum from the first plurality of photonsand an intrinsic calibrant spectrum from the second plurality ofphotons; comparing the intrinsic calibrant spectrum with a referencespectrum for said intrinsic calibrant to determine a wavelength-shift inthe calibrant spectrum; applying the wavelength-shift to the samplespectrum to obtain a calibrated sample

In another embodiment, the disclosure relates to a system forsimultaneous calibration and spectral imaging of a sample, the systemcomprising: an optical train containing an intrinsic calibrant andhaving a first optical path and a second optical path, the first opticalpath simultaneously illuminating the sample and the intrinsic calibrantwith a plurality of illuminating photons and a second optical pathcollecting a first plurality of photons from the sample and a secondplurality of photons from the intrinsic calibrant; a spectrograph forforming a sample spectrum from the first plurality of photons and anintrinsic calibrant spectrum from the second plurality of photons; afirst processing circuitry for comparing the intrinsic calibrantspectrum with a reference spectrum for the intrinsic calibrant todetermine a wavelength-shift; and a second processing circuitry forobtaining a calibrated sample spectrum by applying the wavelength-shiftto the sample spectrum.

BRIEF DESCRIPTION OF THE DRAWINGS

Exemplary embodiments of the disclosure will be illustrated withreference to the following non-limiting illustrations in which:

FIG. 1 is an exemplary algorithm according to one embodiment of thedisclosure;

FIG. 2 is an exemplary auto-calibration system according to oneembodiment of the disclosure;

FIG. 3 is an exemplary auto-calibration system according to anotherembodiment of the disclosure;

FIG. 4 is a representative spectrometer in accordance with an embodimentof the disclosure;

FIG. 5 shows dispersive Raman spectra of a calibrant and a sampleaccording to one embodiment of the disclosure; and

FIG. 6 shows filtered (smoothed) dispersive Raman spectra for thecalibrant and the sample of FIG. 5.

DETAILED DESCRIPTION

The disclosure generally relates to a method and apparatus for automatedspectral calibration of a spectroscopic device, The spectroscopic devicecan be used, for example, for Raman spectroscopy, visible absorptionspectroscopy, near infrared absorption spectroscopy, infrared absorptionspectroscopy, fluorescence spectroscopy or a combination thereof.Further, the spectroscopic device can include a conventionalspectrometer configured according to the embodiments disclosed herein.

FIG. 1 is an exemplary algorithm according to one embodiment of thedisclosure. In step 110 of algorithm 100, both the sample and thecalibrant are illuminated simultaneously. The calibrant can be anyreference material whose spectrum at a given wavelength or wave-numberis known. The term “wavelength” or “wave-number” may apply tofluorescence, near-IR, IR or visible spectroscopy, but wavelength-shiftor wave-number shift (interchangeably, Raman shift) conventionallyapplies to Raman spectroscopy. A non-exhaustive list of calibrantsincludes: acetaminophen, polymethyl methacrylate (PA), oxygen, nitrogen,neon, krypton, xenon, BK-7 glass, quartz, fused silica, naphthalene, 1,4bis (2-methylstyryl) benzene, sulfur, toluene, acetonitrile,benzonitrile, cyclohexane, polystyrene, dysprosium oxide, and holmiumoxide. Because the calibrant's spectrum at a given wavelength is known,it can be used as a baseline reference to calibrate the spectrometer.

The sample can be a chemical, a biological or any substance whoseidentity is detectable by spectroscopic analysis. Once illuminated, insteps 120 and 130 a preliminary spectrum of the sample and the calibrantcan be obtained. Steps 120 and 130 can be implemented simultaneously orsequentially. In step 140, a reference spectrum for the calibrant isobtained having well-known peak position (i.e., wavelength) values.

As will be discussed below, each of the reference and measured spectraof the calibrant can include a plurality of intensity peaks. Acomparison of the location of the peaks in the reference spectrum withthe corresponding peaks in the calibrant's measured spectrum can reveala wavelength-shift as shown in step 150.

In step 160 the wavelength-shift is applied to the preliminary samplespectrum. By adjusting the preliminary sample spectrum 120 commensuratewith the wavelength-shift 150, a calibrated sample spectrum 170 can beobtained. As stated, steps 120 and 130 can be implemented simultaneouslyor sequentially. In an embodiment of the disclosure, steps 130-150 areimplemented followed by steps 120, 160 and 170.

FIG. 2 is an exemplary auto-calibration system according to oneembodiment of the disclosure. System 200 of FIG. 2 includes, amongothers, illumination source 210, calibrant 220, sample 230, opticaltrain 240 and spectrometer 250. Illumination source 210 can be anysource appropriate for making a Raman, fluorescence, visibleabsorption/reflectance, infrared (IR) absorption/reflectance and/or nearIR absorption/reflectance measurement.

Illumination source 210 directs photons 211 to optical splitter 212which can direct a first group of the illuminating photons 216 to sample230 by way of mirror or beam splitting element 241. Optical splitter 212can also direct a second group of illuminating photons 213 to calibrant220. In one embodiment illuminating photons 212 are directed through anoptical medium, such as optical fiber 214. In another embodiment,illuminating photons can be filtered through optical lens 215 prior toilluminating calibrant 224. In still another embodiment (not shown),optical lens 215 is positioned to only filter photons 224 collected fromcalibrant 220.

Depending on the illumination properties, photons 224 interacting withcalibrant 220 can be reflected, refracted, emitted, scattered,transmitted and/or absorbed by the calibrant. Collected photons 224 canoptionally be directed to spectrometer 250 through optical fiber 222. Inone embodiment, one or more optical fibers can carry light from thecalibrant to the spectrometer and one or more optical fibers can carrylight from the sample to the spectrometer. The fibers can be inserted inparallel into the entrance slit of the spectrograph. Spectrometer 250can form a preliminary calibrant spectrum for comparison with areference calibrant spectrum.

Similarly, illuminating photons 216 can be directed to mirror 241 forilluminating sample 230 with photons 217. Sample illumination can bedirect (not shown) or it can take place through optical train 240.Collected photons 232 from sample 230 can be directed to spectrometer250 through gathering optical train 240 and mirror 242. Optical train240 can have one or more optical filters (not shown) for removingphotons of undesired wavelength from group of photons collected fromsample 232. In addition, photons collected from the sample 232 can bephotons that are reflected, refracted, emitted, scattered, transmitted,and absorbed.

As stated, photons 244 and 224 can be directed to spectrometer 250through fiber optic medium (not shown). Such medium can comprise aplurality of optical fibers assembled for communication withspectrometer 250. In one embodiment, the optical fiber can define abifurcated fiber optic line having a first optical medium and a secondoptical medium positioned adjacent to one another. Thus, a first opticalmedium communicates photons 244 collected from the sample tospectrometer 250 while the second optical medium independentlycommunicates photons 224 collected from the calibrant.

Once photons 224 are received at spectrometer 250, a preliminarycalibrant spectrum can be formed. The preliminary calibrant spectrum canbe compared with a reference calibrant spectrum to determine awavelength-shift as discussed in relation to FIG. 1. In one embodiment,spectrometer 250 and illumination source 210 communicate theillumination wavelength 251 to each other.

Spectrometer 250, having received photons 244 collected from sample 230can form a preliminary sample spectrum. Applying the wavelength-shift tothe preliminary sample spectrum, can result in a calibratedspectrum/image for sample 230. The operation of an exemplaryspectrometer will be discussed further below.

FIG. 3 is an exemplary auto-calibration system according to anotherembodiment of the disclosure. In FIG. 3, system 300 includes, amongothers, illumination source 310, calibrant 320, sample 330, opticaltrain 340 and spectrometer 350. Optical train 340 is configured toreceive calibrant 320 to largely eliminate the need for a separatecalibrant illumination circuit. Referring to FIG. 3, illuminatingphotons 311 are directed 316 through mirror or beam splitting element312 and through optical medium 314. Splitting optics 341 directs a firstplurality of illuminating photons 318 to sample 330 while directing asecond plurality of illuminating photons 317 to calibrant 320.

In one embodiment, optical train 340 is configured with a compartmentfor receiving calibrant 320. Such compartment can be devised to provideeasy access to calibrant 320. For example, if a solid calibrant such asacetaminophen is used, optical train 340 can be configured to have acompartment for receiving calibrant 320. Depending on the nature of thecalibrant, it may also be composed of or coated on an optical lens ofoptical train 340. For example, if the calibrant is a polymer materialsuch as PMMA, it can be composed of or coated on a lens of optical train340. Such materials can be used to automatically calibrate thespectrometer without sacrificing the ability to make a spectroscopicmeasurement.

Photons 322 collected from the calibrant 320 can be directed tospectrometer 350 along with photons 332, 344 collected from the sample332. One or more optical mediums (not shown) and mirrors orbeam-splitters 345 can be configured to communicate the collectedphotons to spectrometer 350. Additional filters and lenses can be usedto further remove photons of unwanted wavelength before delivering thecollected photons to spectrometer 350. Spectrometer 350 can form apreliminary sample spectrum and a preliminary calibrant spectrum (notshown). A wavelength-shift can be determined by comparing thepreliminary calibrant spectrum with a reference spectrum for thecalibrant. Once determined, the wavelength-shift can be applied to thesample spectrum to determine a calibrated sample spectrum for sample330.

FIG. 4 is a representative spectrometer in accordance with an embodimentof the disclosure. In FIG. 4, system 400 receives photons collected fromthe sample 414 and photons collected from the calibrant 412 atspectrograph 420. Spectrograph 420 forms a preliminary calibrantspectrum 422 and a preliminary sample spectrum 424. The preliminarysample spectrum 422 can be directed to first processor 430. Processor430 also receives a reference spectrum 452 from database 450. Bycomparing reference spectrum 452 with preliminary calibrant spectrum422, processor 430 can determine a wavelength-shift.

First processor 430 communicates wavelength-shift to second processor440. Second processor 440, having received sample spectrum 440 fromspectrograph 420, can apply the wavelength shift 442 to sample spectrumto obtain a calibrated sample spectrum. The calibrated sample spectrumcan be reported 444 from spectrometer 400.

It should be noted that FIG. 4 provides an exemplary functionalrepresentation and is not intended to limit the scope of the disclosure.For example, the detector associated with spectrograph 420 cancommunicate the sample- or calibrant-collected photons (414, 412,respectively) to a central processing unit via an analog-to-digitalconverter. The detector may be a photodiode array, a charge-coupleddevice (CCD) or a focal plane array. Spectrograph 420 may alternativelybe a liquid crystal tunable filter (LCTF) or similar device coupled withone or more of the for mentioned detector types for spectroscopicimaging. Such devices can operate in connection with a software programapplying the wavelength-shift to the preliminary spectra to obtaincalibrated spectra.

Further, first processor 430 and second processor 440 can be combinedinto one processor (not shown) under the control of a softwareapplication (not shown). Alternatively, the first and second processorscan define one or more firmware devices. Database 450 can be a look-uptable stored in a memory (not shown). The processors and the memory cancontrol optical device such as a photodiode array, a CCD or an LCTF.Further, the optical device can provide a spatially accuratewavelength-resolved image of the sample showing a first and a secondspatial dimension. A spatially accurate wavelength-resolved image is animage of a sample that is formed from multiple “frames” wherein eachframe has plural spatial dimensions and is created from photons of aparticular wavelength (or wave number) or from photons in a particularwavelength band (or wave number band) so that the frames may be combinedto form a complete image across all wavelengths (wave numbers) ofinterest.

As discussed, suitable polymeric calibrants (e.g., PMMA) can be coateddirectly on a lens (or a portion of the lens) of the gathering optics toprovide means for automatic calibration. The gathering lens can be partof the optical train associated with the spectrometer. Coating thecalibrant on the gathering lens does not necessarily inhibit one'sability to acquire a spectrum of the sample.

FIG. 5 shows a dispersive Raman spectroscopy of a calibrant and a sampleaccording to one embodiment of the disclosure. Referring to FIG. 5,spectrum 510 is the Raman spectrum for calibrant PMMA. Spectrum 510shows Raman intensity peaks 512, 513, 514, 515 and 516, each of whichhas been wavelength-shifted to account for the system calibration.Spectrum 520 represent sample spectrum for Bacillus Globigii (BG)spores. Bacillus globigii spores have been used as an anthrax simulant.Spectrum 520 shows Raman intensity peaks 521, 522, 523 and 524. Each ofspectra 510 and 520 were collected using gathering optics which was notcoated with PMMA.

Spectrum 530 shows BG spectrum collected while having PMMA in theexcitation/collection optical path. Spectrum 530 shows Raman intensitypeaks 531, 532, 533 and 534. As seen in FIG. 5, peaks 531, 532, 533 and534 are substantially at the same Raman wavelength shift as thecorresponding Raman intensity peaks of spectrum 520. It is evident fromFIG. 5 that coating one or more gathering lens of an optical train doesnot impair the ability to collect a recognizable sample's spectrum.

FIG. 6 shows a filtered (smoothed) dispersive Raman spectra for thecalibrant and the sample of FIG. 5. FIG. 5 has been filtered to improvethe signal-to-noise ratio of the data. Raman intensity peaks 612, 613,614, 615 and 616 of spectrum 610 are consistent with the respectiveRaman intensity peaks of spectrometer 510. Similarly, Raman intensitypeaks 621, 622, 623 and 624 are consistent with Raman intensity peaks521, 522, 523 and 524, respectively, of spectrum 520. Finally, Ramanspectrum 630 is consistent with unfiltered Raman spectrum 530 as each ofRaman intensity peaks 631, 632, 633 and 634 corresponds with Ramanintensity peaks 531, 532, 533 and 534. The filtered spectra of FIG. 6further show that coating an optical lens with a calibrant will notimpair the ability to collect a recognizable sample's spectrum.

The above description is not intended and should not be construed to belimited to the examples given but should be granted the full breadth ofprotection afforded by the appended claims and equivalents thereto.Although the disclosure is described using illustrative embodimentsprovided herein, it should be understood that the principles of thedisclosure are not limited thereto and may include modification theretoand permutations thereof.

1. A method for simultaneous calibration and spectral imaging of asample comprising: simultaneously illuminating the sample and anintrinsic calibrant with a plurality of illuminating photons from asingle source; receiving, at a spectrometer, a first plurality ofphotons collected from the sample and a second plurality of photonscollected from the calibrant; forming a calibrant spectrum from thesecond plurality of collected photons and a sample spectrum from thefirst plurality of collected photons; comparing the calibrant spectrumwith a reference spectrum of the calibrant to determine awavelength-shift in the calibrant spectrum; and applying thewavelength-shift to the sample spectrum to obtain a calibrated samplespectrum.
 2. The method of claim 1, wherein the photons collected fromthe sample are selected from the group consisting of photons reflected,refracted, emitted, scattered, transmitted and absorbed by the sample.3. The method of claim 1, wherein the sample spectrum is one of a Ramanspectrum, visible absorption spectrum, near infrared absorptionspectrum, infrared absorption spectrum or fluorescence spectrum.
 4. Themethod of claim 1, wherein the calibrant is selected from the groupconsisting of acetaminophen, polymethyl methacrylate, oxygen andnitrogen, neon, krypton, xenon, BK-7 glass, quartz, fused silica,naphthalene, 1,4 bis (2-methylstyryl) benzene, sulfur, toluene,acetonitrile, benzonitrile, cyclohexane, polystyrene, dysprosium oxide,and holmium oxide.
 5. The method of claim 1, wherein the spectrometer isa dispersive spectrometer.
 6. The method of claim 1, wherein thespectrometer is an imaging spectrometer.
 7. The method of claim 6,wherein the imaging spectrometer includes a liquid crystal tunablefilter.
 8. The method of claim 1, wherein the plurality of illuminatingphotons have a first wavelength.
 9. The method of claim 1, wherein thestep of receiving the plurality of photons collected from the sample andthe plurality of photons collected from the calibrant further comprisesseparating the photons collected from the sample from the photonscollected from the calibrant.
 10. The method of claim 1, wherein thestep of receiving the plurality of photons collected from the sample andthe plurality of photons collected from the calibrant further comprisesfiltering the received photons to remove photons of an undesiredwavelength.
 11. A system for simultaneous calibration and dispersiveand/or spectral imaging of a sample comprising: an input forsimultaneously receiving a first plurality of photons collected from thesample and a second plurality of photons collected from an intrinsiccalibrant, wherein said first plurality of photons and said secondplurality of photons originate from the same source; a spectrometer forforming a sample spectrum from the first plurality of photons and acalibrant spectrum from the second plurality of photons; a firstprocessor for comparing the calibrant spectrum with a reference spectrumof the calibrant to determine a wavelength-shift in the calibrantspectrum; and a second processor for applying the wavelength-shift tothe sample spectrum to obtain a calibrated sample spectrum.
 12. Thesystem of claim 11, wherein the photons collected from the sample areselected from the group consisting of photons reflected, refracted,luminesced, fluoresced, Raman scattered, transmitted, absorbed, andemitted by the sample.
 13. The system of claim 11, further comprising anillumination source for simultaneously illuminating the sample and thecalibrant with a plurality of illuminating photons.
 14. The system ofclaim 11, wherein the first processor and the second processor defineone processor.
 15. The system of claim 11, further comprising a memoryfor storing and communicating the reference spectrum of the calibrant tothe first processor.
 16. The system of claim 11, wherein the firstprocessor is programmed with instructions to: a) determine a pluralityof peak locations in the calibrant spectrum; b) determine a plurality ofpeak locations in the reference spectrum of the calibrant; c) comparethe plurality of peak locations in the calibrant spectrum with aplurality of corresponding peak locations in the reference spectrum ofthe calibrant; and d) determine the wavelength-shift as a function ofthe comparison between at least one peak location in the calibrantspectrum and a corresponding peak location in the reference spectrum.17. The system Of claim 11, wherein the second processor is programmedwith instructions to: a) receive the sample spectrum from thespectrograph; b) receive the wavelength-shift from the first processor;and b) calibrate the sample spectrum as a function of thewavelength-shift.
 18. The system of claim 11, wherein the samplespectrum is one of a Raman spectrum, visible absorption spectrum, nearinfrared absorption spectrum, infrared absorption spectrum orfluorescence spectrum.
 19. The system of claim 11, wherein the calibrantis selected from the group consisting of acetaminophen, polymethylmethacrylate, oxygen and nitrogen, neon, krypton, xenon, BK-7 glass,quartz, fused silica, naphthalene, 1,4 bis (2-methylstyryl) benzene,sulfur, toluene, acetonitrile, benzonitrile, cyclohexane, polystyrene,dysprosium oxide, and holmium oxide.
 20. The system of claim 11, whereinthe spectrometer is a dispersive spectrometer.
 21. The system of claim11, wherein the spectrometer is an imaging spectrometer.
 22. The systemof claim 21, wherein the imaging spectrometer includes a liquid crystaltunable filter.
 23. The system of claim 13, wherein the illuminatingphotons have a first wavelength.
 24. The system of claim 11, furthercomprising a medium for communicating the first plurality of photonscollected from the sample and the second plurality of photons collectedfrom the calibrant.
 25. The system of claim 24, wherein the medium is anoptical fiber.
 26. The system of claim 11, further comprising a firstmedium for communicating the first plurality of photons collected fromthe sample and a second medium for communicating the second plurality ofphotons collected from the calibrant.
 27. (canceled)
 28. An apparatusfor simultaneous calibration and dispersive and/or spectral imageacquisition of a sample, comprising: a processing circuit forsimultaneously receiving a calibrant spectrum and a sample spectrumwherein the calibrant is an intrinsic calibrant and further wherein saidcalibrant spectrum and said sample spectrum are formed using photonsfrom a single source; and a memory in communication with the processingcircuit, the memory storing instructions for the processing circuit to:process the calibrant spectrum to locate and identify a plurality ofpeaks, compare the plurality of peak locations in the calibrant spectrumwith a plurality of corresponding peak locations in a reference spectrumof the calibrant, and determine a wavelength-shift as a function of acomparison between at least one peak location in the calibrant spectrumand a corresponding peak location in the reference spectrum; andcalibrate the sample spectrum by applying the wavelength-shift to thesample spectrum.
 29. The apparatus of claim 28, wherein applying thewavelength-shift further comprises: (a) identifying a plurality of peaklocations in the sample spectrum; and (b) applying the wavelength-shiftto at least one of the plurality of peak locations in the samplespectrum.
 30. The apparatus of claim 28, further comprising a databasefor storing a reference spectrum of the calibrant.
 31. The apparatus ofclaim 28, wherein the processing circuit comprises at least onemicroprocessor.
 32. The apparatus of claim 28, wherein the apparatus isa spectrometer.
 33. The apparatus of claim 28, wherein the samplespectrum is one of a Raman spectrum, visible absorption spectrum, nearinfrared absorption spectrum, infrared absorption spectrum orfluorescence spectrum.
 34. The apparatus of claim 28, wherein thecalibrant is selected from the group consisting of acetaminophen,polymethyl methacrylate, oxygen and nitrogen, neon, krypton, xenon, BK-7glass, quartz, fused silica, naphthalene, 1,4 bis (2-methylstyryl)benzene, sulfur, toluene, acetonitrile, benzonitrile, cyclohexane,polystyrene, dysprosium oxide, and holmium oxide.
 35. The apparatus ofclaim 32, wherein the spectrometer is a dispersive spectrometer.
 36. Theapparatus of claim 32, wherein the spectrometer is an imagingspectrometer.
 37. The apparatus of claim 36, wherein the imagingspectrometer includes a liquid crystal tunable filter.
 38. A method forsimultaneous calibration and imaging of a sample in a spectrometer, themethod comprising: simultaneously illuminating the sample and anintrinsic calibrant with a plurality of illuminating photons from asingle source; receiving, at a spectrometer, a first plurality ofphotons collected from the sample and a second plurality of photonscollected from the intrinsic calibrant; forming a sample spectrum fromthe first plurality of photons and an intrinsic calibrant spectrum fromthe second plurality of photons; comparing the intrinsic calibrantspectrum with a reference spectrum for said intrinsic calibrant todetermine a wavelength-shift in the calibrant spectrum; applying thewavelength-shift to the sample spectrum to obtain a calibrated samplespectrum.
 39. The method of claim 38, wherein imaging defines at leastone of obtaining dispersive spectral data or spectral imaging data fromthe sample.
 40. The method of claim 38, wherein the first plurality ofphotons collected from the sample are selected from the group consistingof photons reflected, refracted, luminescence-emitted,fluorescence-emitted, Raman scattered, transmitted, absorbed, andemitted by the sample.
 41. The method of claim 38, wherein the intrinsiccalibrant is selected from the group consisting of acetaminophen,polymethyl methacrylate, oxygen and nitrogen, neon, krypton, xenon, BK-7glass, quartz, fused silica, naphthalene, 1,4 bis (2-methylstyryl)benzene, sulfur, toluene, acetonitrile, benzonitrile, cyclohexane,polystyrene, dysprosium oxide, and holmium oxide.
 42. The method ofclaim 38, wherein the sample spectrum is one of a Raman spectrum, nearinfrared absorption/reflectance spectrum, infraredabsorption/reflectance spectrum, visible absorption/reflectance spectrumor fluorescence spectrum.
 43. The method of claim 38, wherein thespectrometer is a dispersive spectrometer.
 44. The method of claim 38,wherein the spectrometer is an imaging spectrometer.
 45. The method ofclaim 44, wherein the imaging spectrometer includes a liquid crystaltunable filter.
 46. The method of claim 38, wherein the plurality ofilluminating photons have a first wavelength.
 47. The method of claim38, wherein the step of receiving the first plurality of photons fromthe sample and the second plurality of photons from the calibrantfurther comprises separating the first plurality of photons from thesecond plurality of photons.
 48. The method of claim 38, wherein thestep of receiving the first plurality of photons from the sample and thesecond plurality of photons from the calibrant further comprisesfiltering the received photons to remove photons of an undesiredwavelength.
 49. A system for simultaneous calibration and spectralimaging of a sample, the system comprising: an optical train containingan intrinsic calibrant and having a first optical path and a secondoptical path, the first optical path simultaneously illuminating thesample and the intrinsic calibrant with a plurality of illuminatingphotons from a single source and the second optical path collecting afirst plurality of photons from the sample and a second plurality ofphotons from the intrinsic calibrant; a spectrograph for forming asample spectrum from the first plurality of photons and an intrinsiccalibrant spectrum from the second plurality of photons; a firstprocessing circuitry for comparing the intrinsic calibrant spectrum witha reference spectrum for the intrinsic calibrant to determine awavelength-shift; and a second processing circuitry for obtaining acalibrated sample spectrum by applying the wavelength-shift to thesample spectrum.
 50. The system of claim 49, wherein the first pluralityof photons are selected from the group consisting of photons reflected,refracted, luminesced, fluoresced, Raman scattered, transmitted,absorbed, and emitted by the sample.
 51. The system of claim 49, furthercomprising an illumination source.
 52. The system of claim 49, whereinthe optical train further comprises an objective lens.
 53. The system ofclaim 52, wherein the intrinsic calibrant is coated on a portion of theobjective lens.
 54. The system of claim 49, wherein the first opticalpath is a fiber optic medium.
 55. The system of claim 49, wherein thesecond optical path defines a bifurcated optical fiber.
 56. The systemof claim 49, wherein the first processing circuitry further comprises atleast one microprocessor in communication with a memory.
 57. The systemof claim 49, wherein the first processing circuitry and the secondprocessing circuitry define a microprocessor.
 58. The system of claim49, wherein the first processing circuitry and the second processingcircuitry define a firmware.
 59. The system of claim 49, wherein thesample spectrum is one of a Raman spectrum, near infraredabsorption/reflectance spectrum, infrared absorption/reflectancespectrum, visible absorption/reflectance spectrum or fluorescencespectrum.
 60. The system of claim 49, wherein the calibrant is selectedfrom the group consisting of acetaminophen, polymethyl methacrylate,oxygen and nitrogen, neon, krypton, xenon, BK-7 glass, quartz, fusedsilica, naphthalene, 1,4 bis (2-methylstyryl) benzene, sulfur, toluene,acetonitrile, benzonitrile, cyclohexane, polystyrene, dysprosium oxide,and holmium oxide.
 61. The system of claim 49, wherein the spectrographis a spectrometer.
 62. The system of claim 49, wherein the plurality ofilluminating photons have a first wavelength.
 63. The system of claim49, further comprising an optical filter for separating the firstplurality of photons from the second plurality of photons.
 64. Thesystem of claim 49, wherein the optical train further comprises anoptical filter for separating the first plurality of photons from thesecond plurality of photons.
 65. The system of claim 49, wherein theintrinsic calibrant is coated on a portion of the optical traininterposed in the first optical path.